Helicobacter pylori (which has previously been known as Campylobacter pylori) is a Gram-negative bacillus discovered from a chronic gastritis biopsy specimen as a spiral bacterium in 1983 [J. R. Warren and B. Marshall, Lancet, i, 1273 (1983)].
After the discovery thereof, the significance of this bacterium to chronic gastritis and duodenal ulcer has attracted attention, and investigation thereof has become one of important themes biologically and medically. In particular, elucidation of the mechanisms of cell injuries leading to diseases and developments of new inspection methods have been gained attention.
Methods for detecting Helicobacter pylori (hereinafter briefly referred to as HP) include cultivating methods, urease tests, expiratory tests, histological inspections and serological methods. In particular, the serological methods, in which anti-HP antibodies in the sera are detected, are excellent in that they are noninvasive and a large amount of specimens can be treated.
Various anti-HP antibodies are contained in the sera, so that measurement sensitivity and specificity thereof vary depending on the preparation methods and kind of antigens used for antibody measurement. As a result, the clinical significance of assaying the anti-HP antibodies are also various.
For example, detection methods using lysates of all HP cells as antigens for detection of the anti-HP antibodies [B. J. Rathbone, Lancet, ii, 1217 (1985)] sometimes cause false positive judgement due to cross reaction antibodies with other Campylobacter, or conversely, false negative judgement caused by interaction between antigens or denaturation in antigen preparation stages.
B. J. Rathbone reports that antigens partially purified with acidic glycine extract from cells are improved in both specificity and sensitivity than lysates of all HP cells [B. J. Rathbone, "Serological Response to Helicobacter pylori", Eur. J. Gastroenterol. Hepatol., 4 (Supple. 1), S21-S23 (1992)]. Furthermore, he reports that methods using urease, etc. highly purified by high performance liquid chromatography (HPLC), etc. as antigens for detection have a tendency to enhance specificity, but to reduce sensitivity because of genetic diversity between HP strains.
Further, of antibodies corresponding to various antigen components of HP, antibodies were screened which were important in relation to utility on serum diagnosis and pathology of gastropathy and duodenal diseases. von Wulffen et al. report that IgG and IgA antibodies are useful, and that particularly, antibodies to 110-KD and 22-KD antigens characteristically appear in the pathology of these diseases [H. von Wulffen, J. Heesemann, G. W. Bulzow et al., J. Clin. Microbiol., 24, 716-720 (1986)]. C. S. Goodwin et al. conduct ELISA using acidic glycine extract as an antigen, and report that main antigen molecules are 84 KD, 33 KD, 28 KD and 25 KD, in addition to 64 KD, 62 KD and 57 KD. [C. S. Goodwin, E. Blincow, G. Peterson et al., J. Infect. Dis., 155, 488-494 (1987)]. Hirschl et al. report that a 128-KD antigen is useful. A. R. Stacey, D. G. Newell et al. fractionate respective antigen components by HPLC, and report that antibodies to HP-derived urease are useful on serum diagnosis [A. R. Stacey, P. R. Hawtin and D. G. Newell, Eur. J. Clin. Microbiol. Infect. Dis., 9, 732-737 (1990)]. D. J. Evans et al. establish ELISA using a 600-KD or more high molecular weight cell-associated protein as an antigen [D. J. Evans Jr., D. G. Evans, D. X. Grahm et al., Gastroenterology, 96, 1004-1008 (1986)].
On the other hand, T. Sugiyama et al. try to detect anti-HP antibodies in the blood by use of HP-derived antigens purified by monoclonal antibodies. As a result, they report that an antigen named "CP2 antigen" (molecular weight: about 60 KD) is excellent in detection specificity of HP, and that the antibody titer thereof has a high correlation with the pathology of gastritis [T. Sugiyama et al., Gastroenterology, 101, 77-83 (1991) and Toshiroh Sugiyama et al., Nippon Shokakibyo Gakkaisi, 85, 1128 (1988)].
In order to actually obtain the HP antigens by the conventional methods, it has hitherto been necessary to isolate the antigens directly from HP cells. However, in order to obtain the HP cells, it is necessary to use expensive liquid media containing 10% horse sera under microaerobic conditions and to conduct culture for a long period of time of about 5 days. Further, the content of the target antigens in all the cells is so small that purification by separation is difficult, resulting in difficulty of obtaining purified antigens in amounts as large as industrially available.
Also for the CP2 antigen which is considered to be particularly useful as a specific antigen, its purification by separation has been difficult and complicated.